Direct Sequencing

Direct sequencing refers to the sequence analysis of PCR products without prior subcloning into sequencing vectors. Direct sequencing is generally accepted as most reliable to scan for new mutations as well as to score for known mutations.

Because of its reliability and low error rate (equal to or less than one in 10,000 bases), the performance of all other analytical methods is traditionally measured against direct (Sanger) sequencing.

Chain termination by dideoxy nucleotides is used to specify the genetic change and precisely defines the location and nature of the alteration.3,55,56 Initially, Sanger used a polyacrylamide gel platform for fractionation of DNA fragments and autoradiography for detection. Slab gel detection has since been superseded by microcapillary electrophoresis41 and with the introduction of fluorescent-based sequencing tools, direct sequencing has been automated for high-throughput applications.57 Since none of the other scanning methods is capable of such precise definition, direct sequencing is the final step to guarantee results obtained by any of the other methods of scanning.

Despite its accuracy and reliability, direct sequencing on a large scale is laborious and expensive. Ideally, the optimal technique for mutation detection would be rapid, could screen large regions of DNA with high sensitivity and specificity, would not require hazardous chemicals or elaborate, expensive equipment, and yet would provide information about the location and nature of the mutation. Numerous procedures have been developed that meet one or more of these conditions, but no procedure is definitive.